Electrochemical methods such as potentiometry or voltammetry have been commonly applied in the analytical laboratories to determine specific chemical components contained in clinical, environmental or industrial samples. However, the samples collected from remote sampling sites often require special pretreatment to prevent them from contamination or degradation during transportation to the laboratories. The laboratory-based instruments is expensive to operate, and should be managed by the technicians specially trained in the area. In addition to the aforementioned problems, some clinical samples, e.g., blood for in vitro tests, would be collected in smaller volume in order to reduce the patient's uncomfortable feelings and shock. For these reasons, on-spot-monitoring or point-of-care-testing (POCT) with convenient and economic hand-held devices is becoming popular to expedite the analytical tasks, while reducing the laboratory operational costs and errors by non-specialists.
To design portable electroanalytical devices, miniaturization of the electrochemical sensor system including reference and working electrodes should be attained. Numerous schemes have been proposed to accomplish miniaturized working electrodes, and many of them are now commercially available, while reference electrodes have not been extensively developed for miniaturization thereof.
An ideal miniaturized reference electrode requires several characteristics, such as insensitive response to the sample's composition change, providing a constant potential which is characteristic of a reversible redox couple reaction within the half cell. The reversibility of a fast redox reaction ensures the recovery of the same potential even after the passage of electric currents through the reference cell. The reference cell including insoluble metal salt (e.g., Ag/AgCl) provides constant potential by exchanging a cation or anion which is normally contained in a high concentration of reference electrolyte, either in aqueous solution or in a hydrogel medium, which is in contact with the sample solution through a junction. Accordingly, when such a reference system is miniaturized, it is necessary to implement a micro junction, easily activated reference electrolyte, and stable insoluble metal salt on a chip. To use the miniaturized reference electrode for an electrochemical POCT system, fast stabilization of the potential and reproducibility are prerequisite.
Several types of miniaturized reference electrodes have been proposed previously. A reference electrode comprising a screen printed silver/silver chloride electrode, a layer of low temperature melting glass paste or a silicone polymer paste containing potassium chloride, and a hydrophobic polymer membrane that can form micro junctions by hydration has been reported (Cranny, A. W. J.; Atkinson, J. K., Meas. Sci. Technol. 1998, 9, 1557-1565). Unfortunately, the presoaking time for this reference system takes more than an hour and the potential is unstable, although it provides long operational lifetime. Another example of small reference electrode was prepared by the following steps: placing an inner reference electrolyte in the form of hydrogel on a silver/silver chloride electrode; and covering the hydrogel layer with a hydrophobic polymer membrane with a small opening (Lauks, I. R., U.S. Pat. No. 4,993,048). This reference electrode system provides a constant potential only for a short period of time (a few minute) as the small volume of inner reference electrolyte depletes into the sample solution through the junction, and changes the inner salt concentration. However, the formation of a small exposure, which is typically a few micrometers, in the process of mounting an outer membrane is not easy and often results in malfunction of the reference electrode system due to the sealed pore. In addition, there is a possibility that large particulates contained in sample may block the junction. Such an abnormality can not be predicted in advance and lowers the reliability of the measurement. Miniaturized reference electrodes based on field effect transistor (FET) were also proposed (Potter, W.; Dumschat, C.; Cammann, K., Anal. Chem., 1995, 67, 4586-4588): the top of the ion-selective layer is coated with another layer containing solid potassium perchlorate. Since potassium perchlorate is not readily soluble in aqueous solutions, the perchlorate activity in the aqueous surface layer is equal to that of a saturated solution and leads to a constant potential. However, to deposit the potassium perchlorate layer, this system requires a well (diameter 2 mm, and height 1.5 mm) affixed on the chip and is not suitable for mass fabrication.
In order to develop planar reference electrodes that are suitable for mass fabrication with stable reference potential for an elongated period of time, porous material such as cotton thread, glass fiber, cellulose nitrate, cellulose acetate, filter paper and any material that can induce capillary action; porous polymer membrane; or viscous inner reference solution enclosed in a well formed with a punched film layer has been provided as the junction of the reference electrode. The present invention discloses that the aforementioned reference electrodes maintain stable potentials and provide a relatively short activation period. Hence, it can be employed both in potentiometry and voltammetry. The reference electrodes proposed in this invention can be easily miniaturized in planar structure and appropriate for mass fabrication.